We have now turned our efforts toward enhancing GVT effects against breast cancer. We have been studying the use of T cells with a type 2 cytokine profile as therapy against breast cancer. These studies were initiated in the Fowler laboratory where it has been demonstrated in murine models that cultured Th2/Tc2 cells abrogate graft rejection, are associated with reduced levels of GVHD, and have activity against breast cancer, all of which are desirable characteristics for clinical use. Based on these murine studies, we have initiated a clinical trial, 04-C-131, for the treatment of metastatic breast cancer utilizing graft engineering with dose-escalated Th2/Tc2 cells in an effort to facilitate rapid lymphoid donor engraftment with reduced GVHD. The objective of this trial is to determine the response to a Th2/Tc2 augmented allograft in patients with advanced metastatic breast cancer. If the objectives of this trial can be achieved, it would provide a useful platform to test novel forms of allogeneic cellular therapy alone or in combination with conventional therapies. B. Enhancement of GVT effects through rapid donor chimerism Non-Hodgkin's lymphomas (NHL) are a significant health problem in North America where more than 55,000 new cases of NHL are diagnosed each year. Both autologous and allogeneic HSCT have efficacy in NHL that either fail to enter into a remission or have recurred after conventional therapy. Relapse is the most common cause of treatment failure after reduced-intensity allogeneic HSCT in this disease. We have hypothesized that the establishment of rapid and complete donor chimerism, using targeted immune depletion, would enhance GVT effects against NHL. We tested this hypothesis in a clinical trial, 03-C-0077, which utilized the targeted immune depletion approach in patients with advanced NHL. We found that the most important prognosis factor was chemo-sensitivity prior to transplant. These studies demonstrated that rapid complete donor chimerism, after reduced-intensity allogeneic stem cell transplantation results in potent and durable GVT effects against advanced NHL. Tumor-derived lymphocytes - Our current efforts are now focused on further enhancement of GVT effects against NHL. The standard therapy for relapsed disease after transplant is infusion of additional allogeneic lymphocytes, most commonly referred to as a donor lymphocyte infusion (DLI). However, DLI have been demonstrated to have limited efficacy against most NHL. One of the major impediments to effective adoptive immunotherapy for cancer has been generating sufficient high-affinity, tumor-specific T cells to achieve a clinical response. We have developed a research effort in the development of tumor infiltrating lymphocytes for treatment of recurrent disease after allogeneic HSCT. This effort is based upon the likelihood that T cells within tumors are likely of donor origin after transplant, suggesting that they have been tumor specific in their homing. It is also possible that these cells are tumor specific in their antigen specificity and killing activity. Finally, it is possible to activate and expand these cells through ex-vivo costimulation. It is our hypothesis that activated tumor-derived lymphocytes (TDL) will have greater efficacy and more tumor specificity than unmanipulated donor lymphocytes. Over the past two years we have been working extensively in collaboration with the Fowler laboratory, the ETIB Pre-Clinical Support Service, the NIH CC DTM Cell Processing Laboratory, and the June laboratory, as well as receiving extensive consultation from the Rosenberg laboratory, to develop a method to generate allogeneic TDL as a novel form of allogeneic cellular therapy. This method includes CD3/CD28 co-stimulation prior to expansion with IL-2. Preclinical testing indicates that T cells can be generated in sufficient numbers for clinical employment from tumor samples. A protocol for the clinical use of allogeneic TDL for treatment of B cell malignancies which have relapsed after alloHSCT received FDA approval in January 2007. This pilot protocol will provide new clinical information on the feasibility and safety of administering ex-vivo co-stimulated/expanded TDL in addition to providing data on characteristics of residual tumor after allogeneic HSCT, the immunophenotypic and functional characteristics of TDL, and identification of GVT antigen candidates. C. Enhancement of GVT effects through vaccines Our other effort in the enhancement of GVT effects involves the use of vaccines with allogeneic HSCT for the treatment of multiple myeloma. There has also been significant interest in the use of reduced-intensity allogeneic HSCT in multiple myeloma due to a significant decrease in treatment-related mortality with this procedure; however it is associated with higher relapse rates as compared with myeloablative conditioning regimens. Interest in the use of vaccines in allogeneic HSCT to enhance GVT effects against myeloma and reduce relapse arose out of research in the Kwak laboratory when they were members of the ETIB. Multiple myeloma, as well as other B cell malignancies such as follicular NHL, synthesizes a single immunoglobulin (Ig) with unique variable regions, referred to idiotypes (Id) in the heavy and light chains. The idiotypic determinants of the surface Ig of myeloma can thus serve as a tumor-specific marker for the malignant clone. It was our hypothesis that the vaccination of normal allogeneic stem cell donors with patient-derived Id-specific vaccine would generate Id-specific immunity and be passively transferred to the recipient at the time of transplant thereby reducing the risk of relapse in patients with multiple myeloma undergoing reduced-intensity allogeneic HSCT. In collaboration with the Kwak laboratory, which continues after their move to the M.D. Anderson Cancer Center (MDACC), we designed a protocol, 00-C-0201, which combines Id vaccines and our targeted immune-depletion approach with allogeneic HSCT. The goal of this protocol is to transfer tumor antigen-specific immunity induced in the stem cell donor to the allogeneic HSCT recipient to reduce relapse. Vaccine is generated from patient-derived Id conjugated to an immunogenic protein carrier, keyhole limpet hemocyanin (Id-KLH). Allogeneic stem cell donors are vaccinated with Id-KLH prior to stem cell donation to study the adoptive transfer of vaccine-primed lymphocytes. Ten patients and their respective donors have been enrolled onto study. Vaccine was successfully generated from all 10 patients, and all 10 donors completed vaccinations without any grade 3 or 4 toxicities. All 10 donors demonstrated evidence of having developed immunity toward KLH. Seven of 10 donors demonstrated Id-specific T cell immunity, and 8 of 10 demonstrated Id-specific B cell immunity. Among the allogeneic HSCT recipients there have been five complete responses and five partial responses. The results of this trial demonstrated that Id-KLH vaccines can be safely given to normal stem cell donors and vaccination of donors with tumor-specific antigen represents a tactic to potentially enhance responses and reduced relapse after allogeneic HSCT